The conserved Notch signaling pathway is critical for cell fate specification and lateral inhibition during development in vertebrates and invertebrates. Notch receptors bind extracellular ligands (e.g. Delta, Serrate and LAG-2) containing a conserved DSL domain that is critical for receptor activation. We have identified a new family of putative Notch ligands in C. elegans with homologs in vertebrates. Unlike canonical Notch ligands, these proteins lack the conserved DSL domain but they share a conserved sequence motif with classical Notch ligands. The first aim of this proposal examines in detail the function of one of these new putative ligands. SEL-14 is required for normal development and preliminary studies suggest that it activates Notch signaling. The role of SEL-14 in Notch signaling will be examined at a molecular and cellular level using genetics, lineage analysis and immunohistochemistry. The developmental role of C. elegans SEL-14 homologs will also be addressed by identification of deletion alleles, expression analysis, characterization of mutant phenotypes, and genetic analysis. The second aim tests the hypothesis that SEL-14 directly interacts with Notch receptors and cooperates with classical DSL ligands in receptor activation. This model suggested by in vivo studies and will be tested in vitro using two-hybrid studies and tissue culture experiments. We will also examine the role of a previously uncharacterized, conserved sequence motif that is found only in SEL-14, C. elegans homologs and classical Notch ligands. Structure/function studies will address the function of this conserved motif in SEL-14 and in classical Notch ligands. Understanding Notch ligands is critical given the essential role of Notch signaling in development, stem cell maintenance, cancer and memory. Defects in Notch signaling and presenilin processing cause numerous diseases. Deltas has been implicated in spondylocostal dysostosis. Mutations in NotchS and Jaggedl cause CADASIL and Alagille syndromes, respectively. These are dominantly inherited disorders associated with stroke and dementia. Notch and APR not only share common processing pathways like presenilins, but recent studies suggest that Notch and APR may both contribute to Alzheimer's disease pathology like presenilins. All of these disorders are poorly understood and no effective treatment is available. We anticipate that understanding ligand regulation of Notch signaling will be a first step in the development of therapies for these and other disorders.